His-RBX1 (full-length),
HA-DDB1 (full-length),
Cullin4A (full-length),
CRBN (full-length)
＞90% by SDS-PAGE
Store at -80ºC. Avoid multiple
freeze-thaw cycles.
Cullin-4A (CUL4A) is a core component of multiple cullin-RING type Ubiquitin ligase complexes that mediate the ubiquitination of proteins involved in cell cycle progression, DNA repair and other processes. In the DCX complex (DDB1-CUL4-X-box), CUL4A serves as a scaffold that organizes the DDB1-X-box substrate recognition subunits with the RBX1 subunit and contributes to catalysis through positioning of the substrate and an E2 ubiquitin-conjugating enzyme. In vivo, the E3 ubiquitin ligase activity of the DCX complex is dependent on neddylation of the CUL4 subunit, though neddylation may be dispensable in some in vitro reactions.
The Cullin4A-RBX1-DDB1-CRBN activity was measured by protein-tracer binary binding assay
using HTRF
technology.
The Cullin4A-RBX1-DDB1-CRBN activity was measured by PROTAC-mediated Ternary
Complex Formation using HTRF
technology. The dose response curve of dBET1 in the Ternary
Assay exhibited a bell-shaped curve, a hook effect and a peak.
The Cullin4A-RBX1-DDB1-CRBN activity was measured by WB analysis for target protein
ubiquitination assay. The Ubiquitinated BRD4(D1+D2) bands increased with time.
Lane M: Precision Plus Protein™ Dual Color Standards (Bio-Rad) (Sample loaded: 5 μl)
Lane 1: Cullin4A-RBX1-DDB1-CRBN complex under reducing condition (Sample loaded: 2 μg)
Lane 2: Cullin4A-RBX1-DDB1-CRBN complex under reducing condition (Sample loaded: 4 μg)
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